Copolymer-1 improvements in compositions of copolymers

ABSTRACT

The present invention relates to an improved composition of copolymer-1 comprising copolymer-1 substantially free of species having a molecular weight of over 40 kilodaltons.

This application is a continuation of application Ser. No. 09/032,334,filed Feb. 27, 1998, U.S. Pat. No. 6,048,898 which is a continuation ofU.S. Ser. No. 08/447,146, filed May 22, 1995, now U.S. Pat. No.5,800,808, which is a continuation-in-part of U.S. Ser. No. 08/344,248,filed Nov. 23, 1994, now abandoned, which is a continuation of U.S. Ser.No. 08/248,037, filed May 24, 1994, now abandoned.

BACKGROUND OF THE INVENTION

Copolymer-1 is a synthetic polypeptide analog of myelin basic protein(MBP), which is a natural component of the myelin sheath. It has beensuggested as a potential therapeutic agent for multiple sclerosis (Eur.J. Immunol. [1971] 1:242; and J. Neurol. Sci. [1977] 31:433). Allreferences cited herein are hereby incorporated by reference in theirentirety. Interest in copolymer-1 as an immunotherapy for multiplesclerosis stems from observations first made in the 1950's that myelincomponents such as MBP prevent or arrest experimental autoinimuneencephalomyelitis (EAE). EAE is a disease resembling multiple sclerosisthat can be induced in susceptible animals.

Copolymer-1 was developed by Drs. Sela, Arnon, and their co-workers atthe Weizmann Institute (Rehovot, Israel). It was shown to suppress EAE(Eur. J. Immunol. [1971] 1:242; U.S. Pat. No. 3,849,550). More recently,copolymer-1 was shown to be beneficial for patients with theexacerbating-remitting form of multiple sclerosis (N. Engl. J. Med.[1987] 317:408). Patients treated with daily injections of copolymer-1had fewer exacerbations and smaller increases in their disability statusthan the control patients.

Copolymer-1 is a mixture of polypeptides composed of alanine, glutamicacid, lysine, and tyrosine in a molar ratio of approximately 6:2:5:1,respectively. It is synthesized by chemically polymerizing the fouramino acids forming products with average molecular weights of 23,000daltons (U.S. Pat. No. 3,849,550).

It is an object of the present invention to provide an improvedcomposition of copolymer-1.

SUMMARY OF THE INVENTION

The present invention relates to a composition of copolymer-1substantially free of species of copolymer-1 having a molecular weightof over 40 kilodaltons (KDa).

The invention further relates to a copolymer-1 having over 75% of itsmolar fraction within the molecular weight range from about 2 KDa toabout 20 KDa.

In addition, the invention relates to a copolymer-1 having an averagemolecular weight of about 4 to about 8.6 KDa.

Moreover, the invention relates to a pharmaceutical composition and amethod for the treatment of multiple sclerosis, using theabove-discussed copolymer-1.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 displays the molecular weight distribution of three batches ofcopolymer-1, showing the proportion of species with molecular weightabove 40 KDa. FIG. 2 shows similar data relating to the molar fraction.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a composition of copolymer-1substantially free of species of copolymer-1 having a molecular weightof over 40 kilodaltons (KDa). Preferably, the composition contains lessthan 5% of species of copolymer-1 having a molecular weight of 40 KDa ormore. More preferably, the composition contains less than 2.5% ofspecies of copolymer-1 having a molecular weight of 40 KDa, or more.

The invention further relates to a copolymer-1 having over 75% of itsmolar fraction within the molecular weight range from about 2 KDa toabout 20 KDa.

In addition, the invention relates to a copolymer-1 having an averagemolecular weight of about 4 to about 8.6 KDa. In particular, theinvention relates to a copolymer-1 having an average molecular weight ofabout 4 to about 8 KDa and a copolymer-1 having an average molecularweight of about 6.25 to about 8.4 KDa.

Copolymer-1, according to the present invention, may be prepared bymethods known in the art, for example, the process disclosed in U.S.Pat. No. 3,849,550, wherein the N-carboxyanhydrides of tyrosine,alanine, y-benzyl glutamate and E-N-trifluoro-acetyllysine arepolymerised at ambient temperature in anhydrous dioxane withdiethylamine as initiator. The deblocking of the y-carboxyl group of theglutamic acid is effected by hydrogen bromide in glacial acetic acid andis followed by the removal of the trifluoroacetyl groups from the lysineresidues by 1M piperidine. For the purposes of the application, theterms “ambient temperature” and “room temperature” should be understoodto mean a temperature ranging from about 20 to about 26° C.

The copolymer-1 with the required molecular weight profile can beobtained either by methods known per se. Such methods includechromatography of copolymer-1 containing high molecular weight speciesand collecting the fractions without the undesired species or by partialacid or enzymatic hydrolysis to remove the high molecular weight specieswith subsequent purification by dialysis or ultrafiltration. A furthermethod to obtain copolymer-1 with the desired molecular weight profileis by preparing the desired species while the amino acids are stillprotected and then obtain the correct species directly upon removing theprotection. The compositions of the present invention may be formulatedby conventional methods known in the art. Preferably, the composition islyophilized and formed into an aqueous solution suitable forsub-cutaneous injection. Alternatively, copolymer-1 may be formulated inany of the forms known in the art for preparing oral, nasal, buccal, orrectal formulations of peptide drugs.

Typically, copolymer-1 is administered daily to patients suffering frommultiple sclerosis at a dosage of 20 mg.

The invention will be exemplified but not necessarily limited by thefollowing Examples.

EXAMPLE 1 Chromatographic Method of Preparation of Low-toxicityCooolymer-1

Two batches of copolymer-1 were prepared according to the methods knownin the art, for example, U.S. Pat. No. 3,849,550.

One batch was then subjected to chromatographic separation, as describedbelow.

A column for gel filtration, FRACTOGEL TSK HW55 (600×26 mm) was preparedin a Superformance 26 Merck cartridge according to the manufacturer'sinstructions. The column was equilibrated with water and acetonesolution was injected for total volume determination. The column wasequilibrated with 0.2M ammonium acetate buffer pH 5.0. 30 ml copolymer-1samples (20 mg/ml, in 0.2M ammonium acetate pH 5.0) were loaded on thecolumn and fractions were collected every 10 minutes. A fraction havingan average molecular weight of 7-8 KDa was isolated between 120-130minutes (Batch A).

Molecular Weight Analysis

UV absorbance at 275 nm was determined in a UVIKON 810spectrophotometer. Samples were diluted to obtain a UV absorbance lowerthan 1 Absorption Unit. The molecular distribution of the 2 batches wasdetermined on a calibrated gel filtration column (Superose 12).

Copolymer-1 batch A was found to have an average molecular weight of 7-8KDa. 2.5% of this batch had a molecular weight above 32 KDa, but nocopolymer-1 species present in this batch had a molecular weight of over40 KDa.

The other batch of copolymer-1 which was not subjected tochromatography, had an average molecular weight of 12 KDa. 2.56 of thebatch had a molecular weight above 42 KDa and 5% of the totalcopolymer-1 species in this batch had a molecular weight of over 40 KDa.

EXAMPLE 2 Toxicity Analysis

A: In Vivo

Three batches of copolymer-1 having an average molecular weight of 7.3and 8.4 KDa (less than 2.5% copolymer-1 species over 40 KDa) and 22 KDa(more than 5% copolymer-1 species over 40 KDa) were subjected to thetoxicity test described below. In each case 5 mice were used in eachexperimental group.

Method

Copolymer-1 was dissolved in distilled water to yield a solution of 2mg/ml of the active ingredient. Each mouse was injected with 0.5 ml ofthe test solution into the lateral tail vein. Mice were observed formortality and relevant clinical signs over a 48 hour period.Observations were recorded 10 minutes, 24 hours and 48 hourspost-injection. If, at the end of 48 hours, all the animals were aliveand no adverse signs had been observed, then the batch was designated“non-toxic”. If, however, one or more of the mice had died or had shownadverse signs, then the batch was designated “toxic”.

The batches with the average molecular weight of 7.3 and 8.4 KDa wereboth designated “non-toxic”, whereas in the batch with the averagemolecular weight of 22 KDa, 3 out of 5 mice had died at the end of 48hours, and it was consequently designated “toxic”.

B: In Vitro

RBL—Degranulation test

I. Introduction

Histamine (or serotonin) release from basophile is an in vitro model forimmediate hypersensitivity. The Rat Basophilic Leukemia cell line(RBL-2H₃) was developed and characterized as a highly sensitive,uniform, easy to maintain in culture and reproducible system (E. L.Basumian, C. Isersky, M. G. Petrino and R. P. Siraganian. Eur. J.Immunol. 11, 317 (1981)). The physiological stimulus for histaminerelease involves binding of the antigen to membrane-bound IgE molecules,resulting in the latter's cross-linking and the consequent triggering ofan intricate biochemical cascade. Beside these physiological,immunoglobulin-mediated triggers, degranulation can be induced bydifferent non-IgE-mediated stimuli. Among these are various peptides andsynthetic polymers, e.g. polylysine (R. P. Siraganian. Trends inPharmacological Sciences, October 432 (1983)). The RBL degranulationtest is, therefore, used in order to screen out those batches ofcopolymer-1 which evoke substantial degranulation and thus might elicitundesirable local and/or svstemtc side effects.

II. Principle of the test method

Rat Basophilic Leukemia cells (RBL-2H₃), are loaded with [³H]-serotonin, followed by incubation with 100 μg of the copolymer-1 to betested. Batches of copolymer-1 which induce non-specific degranulation,release [³H] -serotonin into the medium. The radioactivity in the mediumis counted by a scintillation counter and the total radiolabeledserotonin incorporated into the cells is determined in the pelletedcells. Percent degranulation is calculated as the percentage ofserotonin released out of the total incorporated.

III. Results

Four batches of copolymer-1, with average molecular weight between6,250-14,500 were analyzed for both % of the species with molecularweight over 40 KDa and for degranulation of RBL's. Results aresummarized in the following table.

Average % of species with % Serotonin M.W. (Daltons) M.W. over 4OKDaRelease  6,250 <2.5 12.4  7,300 <2.5 21.0 13,000 >5 66.9 14,500 >5 67.8

As can be seen, when the % of high molecular weight species is low(<2.5), the % release of serotonin, indicative of toxicity, is low, andvice versa.

EXAMPLE 3 Preparation of Trifluoroacetyl-Copolyrmer-1

Protected copolymer-1 is prepared as described by Teitelbaum et al. Eur.J. Immun. Vol. 1 p. 242 (1971) from the N-carboxyanhydrides of tyrosine(18 g), alanine (50 g), y-benizyl glutamate (35 g) andtrifluoroacetyllysine (83 g) dissolved in 3.5 liters of dioxane.

The polymerization process is initiated by the addition of 0.01-0.02%diethylamine. The reaction mixture is stirred at room temperature for 24hours and then poured into 10 liters water. The product (protectedcopolymer-1) is filtered, washed with water and dried. The removal ofthe gamma-benzyl blocking groups from the glutamate residue is carriedout by treating the protected copolymer-1 with 33% hydrobromic acid inglacial acetic acid at room temperature for 6-12 hours with stirring.The product is poured into excess water, filtered, washed and dried,yielding the trifluoroacetyl-copolymer-1.

EXAMPLE 4 Preoaration of Trifluoroacetyl-Copolymer-1

Protected copolymer-1 is prepared as described by Teitelbaum et al. Eur.J. Immun. Vol. 1 p. 242 (1971) from the N-carboxyanhydrides of tyrosine(18 g), alanine (50 g), τ-benzyl glutamate (35 g) andtrifluoroacetyllysine (83 g) dissolved in 3.5 liters of dioxane.

The polymerization process is initiated by the addition of 0.01-0.02%diethylamine. The reaction mixture is stirred at room temperature for 24hours and then poured into 10 liters water. The product (protectedcopolymer-1) is filtered, washed with water and dried. Protectedcopolymer-1 is treated with 33% HBr in acetic acid which removes theomega benzyl protecting group from the 5-carboxylate of the glutamateresidue and cleaves the polymer to smaller polypeptides. The time neededfor obtaining copolymer-1 of molecular weight 7,000±2,000 Da depends onthe reaction temperature and the size of protected copolymer-1. Attemperatures of between 20-28° C. a test reaction is performed on everybatch at different time periods for example, from 10-50 hours.

The results concerning the molecular weights of these small scalereactions are calculated and a curve of molecular weight against time isdrawn. The time needed for obtaining molecular weight 7,000±2,000 Da iscalculated from the curve and performed on larger scale reaction. Onaverage, working at 26° C. the time period is 17 hours. The product ispoured into excess water, filtered, washed and dried, yielding thetrifluoroacetylcopolymer-1.

Preparation of Low-toxicity Copolymer-1

20 g of trifluoroacetyl-copolymer-1 are dispersed in 1 liter of water towhich 100 g piperidine are added. The mixture is stirred for 24 hours atroom temperature and filtered. The solution of crude copolymer-1 isdistributed into dialysis bags and dialyzed at 10°-20° C. against wateruntil a pH=8 is attained. It is then dialyzed against about 0.3% aceticacid and again water until a pH=5.5-6.0 is obtained. This solution isthen concentrated and lyophilized to dryness.

What is claimed is:
 1. A method for treating multiple sclerosis,comprising administering to a subject in need thereof, apharmaceutically effective amount of a copolymer-1 fraction wherein saidfraction contains less than 5% of species of copolymer-1 having amolecular weight of over 40 kilodaltons; and wherein over 75% of saidcopolymer-1 in said fraction is within a molecular weight range of about2 kilodaltons to about 20 kilodaltons, wherein said copolymer-1 fractionis prepared by a process comprising the steps of: reacting protectedcopolymer-1 with hydrobromic acid to form trifluoroacetyl copolymer-1having over 75% of its molar fraction within the molecular weight rangefrom about 2 kDa to about 20 kDa, wherein said reaction takes place fora time and at a temperature predetermined by test reaction, and treatingsaid trifluoroacetyl copolymer-1 having over 75% of its molar fractionwithin the molecular weight range from about 2 kDa to about 20 kDa withaqueous piperidine solution to form copolymer-1 having over 75% of itsmolar fraction within the molecular weight range from about 2 kDa toabout 20 kDa.